Programmable Single and Multiplex Base-Editing in Bombyx mori Using RNA-Guided Cytidine Deaminases

Abstract

Genome editing using standard tools (ZFN, TALEN, and CRISPR/Cas9) rely on double strand breaks to edit the genome. A series of new CRISPR tools that convert cytidine to thymine (C to T) without the requirement for DNA double-strand breaks was developed recently and quickly applied in a variety of organisms. Here, we demonstrate that CRISPR/Cas9-dependent base editor (BE3) converts C to T with a high frequency in the invertebrate Bombyx mori silkworm. Using BE3 as a knock-out tool, we inactivated exogenous and endogenous genes through base-editing-induced nonsense mutations with an efficiency of up to 66.2%. Furthermore, genome-scale analysis showed that 96.5% of B. mori genes have one or more targetable sites that can be edited by BE3 for inactivation, with a median of 11 sites per gene. The editing window of BE3 reached up to 13 bases (from C1 to C13 in the range of gRNA) in B. mori. Notably, up to 14 bases were substituted simultaneously in a single DNA molecule, with a low indel frequency of 0.6%, when 32 gRNAs were co-transfected. Collectively, our data show for the first time that RNA-guided cytidine deaminases are capable of programmable single and multiplex base editing in an invertebrate model.

Keywords

• CRISPR/Cas9
• base editing
• DSB-free
• Bombyx mori
• multiplex editing